Genetically modified sugarbeet

ABSTRACT

The present invention relates to methods for reducing glutamine metabolism in sugarbeet.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation of application Ser. No. 09/786,534, filedJun. 26, 2001, which is based upon PCT International Application No.PCT/EP99/06522, filed Oct. 17, 2000, claiming priority of GermanApplication No. 198 40 964.8, filed Sep. 8, 1998.

DESCRIPTION

[0002] The present invention relates to the nucleotide sequence of aglutamine synthetase from sugarbeet, to a vector comprising thisnucleotide sequence, to cells which are transformed with this vector, toproteins encoded by this nucleotide sequence, to plants which have beentransformed with this nucleotide sequence, and to methods for thegenetic modification of plants, in particular sugarbeet.

[0003] The accumulation of glutamine as the main α-amino-N componentwhich occurs in the storage root, that is to say the storage organ ofthe sugarbeet, and which is also referred to as harmful nitrogen, givesrise to considerable problems in sugar production. The compensation,generally by rendering alkaline, for the acidification of the beet juicewhich is caused by this component is costly, leads to faster wear on theproduction systems and, finally, also involves considerableenvironmental pollution, which can likewise be prevented only by use ofcostly measures. This glutamine is synthesized in the plant throughamidation of glutamic acid by NH₄ ⁺ being bound, with consumption ofATP, to C-4 of glutamate. This step is catalyzed by the enzyme glutaminesynthetase (abbreviated to GS hereinafter). This enzyme is present bothin the chloroplast and in the cytoplasm of the plant cell. In thechloroplast, the enzyme occurs as a tetramer which is encoded by onegene and consists of up to five subunits (GS-2). In the cytoplasm,according to current knowledge, the enzyme normally occurs as aheterooligomer (GS-1) encoded by more than one gene. The various GS-1isoenzymes known are usually heterooctamers. Six different isoforms ofGS-1 have been found to date. The glutamine synthetase localized in thechloroplast, that is to say GS-2, has the main function of binding theNH₄ ⁺ produced during photorespiration and converting the NH₄ ⁺ derivedfrom nitrate reduction into organic compounds. The function of GS-1 is,by contrast, mainly in catabolic degradation pathways during the courseof which the NH₄ ⁺ resulting from protein degradation is fixed. Suchdegradation pathways are particularly important during aging of theleaf, that is to say during senescence, and the resulting glutamine isexported from the leaf into storage organs.

[0004] In contrast to GS-2 in the chloroplast, relatively little isknown about GS-1 in the cytoplasm. This relates in particular to itsfunction in the cell, but also to its regulation. In contrast to GS-2,it has been assumed to date that GS-1 is encoded by more than one gene,the number thereof being variable. The genes show homologies with oneanother but can be unambiguously separated from one another (Brears etal., Plant J. 1 (1991), 235 to 244; Edwards et at al., Plant Cell 1(1989), 241 to 248). In addition, the GS-1 genes, which each encode onesubunit of the octameric holoenzyme, appear to be regulated differently(Petermann and Goodman MGG 230 (1991), 145 to 154). Controlled externalinfluencing of glutamine metabolism by modification of GS-1 is madedifficult thereby.

[0005] The localization of GS-1 in the plant is also still substantiallyunknown. It is known from Edwards et al. (loc. cit.) that one isoform isthought to be expressed exclusively in the phloem, where it possiblyplays a part in intercellular transport. Sakurai et al. (Planta 200(1996), 306 to 311) reports that in rice one isoform of GS-1 is presentin the conducting bundles and evidently plays a part in the export ofglutamine from leaves. It is also known that tobacco and alfalfa plantswhich have been transformed with a GS-1 gene from Lotus corniculatusshow expression in the flowers (Carrayol et al., Plant Sci 125 (1997),75 to 85). It is additionally known that the composition andlocalization of GS-1 holoenzymes in the root nodules of Lotuscorniculatus may vary greatly.

[0006] No targeted reduction, carried out by methods of molecularbiology, in the glutamine content in plants, especially in storageorgans of plants, has been disclosed to date. The essential difficultiesoccurring in the targeted reduction of the glutamine synthetase activityin plants and the eventually desired reduction in the nitrogen contentin storage organs of plants derive from the fact that the glutaminesynthetase activity must be restricted tissue- and time-specifically insuch a way that the nitrogen content in the target organ, for examplethe storage root of a sugarbeet, is reduced. This must not involve anyimpairment of the other functions and properties of the sugarbeet. Onthe contrary, it must be ensured that the overall physiology of thesugarbeet remains intact and there is merely a specific reduction in thenitrogen content in the storage organ of the sugarbeet. Because of theproblems described concerning the localization of GS-1 and the lack ofclarity in relation to the time specificity of its activity, nosuccessful experiments have been disclosed indicating that it waspossible to reduce the nitrogen content in storage organs of sugarbeetvia modulation of the GS-1 activity.

[0007] The technical problem on which the present invention is based isthus to provide means and methods which make it possible in a targetedmanner, that is to say tissue- and time-specifically, to reduce thenitrogen content in the storage organ of a plant, in particular of asugarbeet, without this involving impairment of the vital, growth andreproductive functions of the sugarbeet and its commercial value.

[0008] The present invention solves this problem by providing anisolated and purified nucleotide sequence and vectors comprising thisnucleotide sequence, which code for one subunit of the GS-1 ofsugarbeet. The present invention solves the technical problem also byproviding the isolated and purified protein encoded by this nucleotidesequence, in particular the amino acid sequence of the GS-1 subunit fromsugarbeet, and by methods for the genetic modification of plants, inparticular sugarbeet, where the content of glutamine synthetase in aplant, in particular in its senescent leaves, is altered, in particularreduced, by transforming cells of this plant with said vectors, andregenerating from the transformed cells intact, propagatable, stablytransformed transgenic plants in whose senescent leaves the activity ofGS-1 is reduced or completely suppressed.

[0009] The invention is surprising and advantageous in particularbecause the subunit encoded by the nucleotide sequences of the inventionrepresents the only subunit of the oligomeric GS-1 isoform occurring inthe senescent leaves of the sugarbeet. Accordingly, the invention alsoprovides the surprising information that this isoform of GS-1 is ahomooctamer. This makes it possible in a surprising manner to influencethe activity of this enzyme by influencing the expression of a singlegene, namely the gene encoding the GS-1 subunit, in particular toprevent or reduce the expression thereof. The invention is alsosurprising inasmuch as the homooligomeric isoform of GS-1 provided bythe invention occurs only in the stage of senescence and accordinglydisplays, besides the tissue specificity in relation to the localizationin the leaf which has been mentioned above, also a time specificity inrelation to the occurrence during senescence. The present inventionsurprisingly therefore provides means and methods for influencing thequalitative and/or quantitative occurrence of a homooctameric GS-1isoform which can be found only in senescent leaves of the sugarbeet.The nucleotide sequences of the invention can also be used for cloninghomologous genes, in particular the coding regions thereof, in othertissues and even other plants and organisms. It is possible inparticular on use of the present nucleotide sequence as hybridizationprobe in homologous or heterologous systems also to identify and isolateendogenous regulatory noncoding nucleotide sequences which areassociated with this sequence and which, for example, mediate time- andtissue-specific expression.

[0010] The invention solves the present technical problem in particularby providing a nucleotide sequence for modulating the expression, inparticular for suppressing the expression, of a protein having theactivity of a glutamine synthetase, in particular the activity of aGS-1, which is selected from the group consisting of

[0011] a) the DNA sequence of SEQ ID No. 1, 3 or a part thereof,

[0012] b) a nucleotide sequence which encodes the amino acid sequence ofSEQ ID No. 2 or a part thereof,

[0013] c) a nucleotide sequence which is complementary to the nucleotidesequences of a) or b), or a part thereof, and

[0014] d) a nucleotide sequence which hybridizes with the nucleotidesequences of a) to c).

[0015] The nucleotide sequence of the invention is functionallycharacterized in that, in a cell transformed therewith and having anendogenous GS-1-encoding sequence, it modulates the activity of the GS-1activity of this transformed cell, for example increases the GS-1activity, for example by overexpression, or reduces or completelysuppresses the GS-1 activity, for example through the nucleotidesequence of the invention being transformed in the form of an antisenseconstruct which inhibits endogenous GS-1 translation.

[0016] The invention provides in a particularly preferred embodiment forthe nucleotide sequence to be derived from the sugarbeet Beta vulgaris.

[0017] The nucleotide sequence of the invention may be a DNA sequence,for example a genomic, where appropriate intron-interrupted DNA sequenceor cDNA sequence, but it can also be an RNA sequence, for example anmRNA sequence or synthetically prepared. The present invention relatesboth to the sense and to the antisense nucleotide sequences. Thenucleotide sequences of the invention can be so-called full-lengthsequences, that is to say sequences which encode a complete proteinhaving the activity of a glutamine synthetase, in particular of the GS-1of sugarbeet, where appropriate including the translation initiationsite. However, the invention also relates to partial sequences of suchnucleotide sequences, in particular those which serve to modulate theexpression of the protein having the activity of a glutamine synthetase,in particular of a GS-1 from sugarbeet. Accordingly, the nucleotidesequence of the invention may also form a fusion gene in a transcriptionor translation unit with other nucleotide sequences. The inventionrelates in an advantageous refinement also to nucleotide sequences whichhybridize with the DNA sequence specified in SEQ ID No. 1 or 3 orhybridize with a nucleotide sequence which encodes the amino acidsequence of SEQ ID No. 2, and nucleotide sequences which hybridize withnucleotide sequences complementary to the two sequences mentioned.

[0018] In connection with the present invention, hybridization means aprehybridization, a hybridization and subsequent washing. Theprehybridization preferably takes place in an aqueous solution composedof 6×SSPE, 0.1% SDS and 5× Denhardt's reagent, and 500 μg/ml denaturedherring sperm at 60° C., particularly preferably at 65° C., for 3 hours.The hybridization preferably takes place in an aqueous solution composedof 3×SSPE, 0.1% SDS, 5× Denhardt's reagent and 500 μg/ml denaturedherring sperm at 60° C., particularly preferably at 65° C., for 16hours. The washing preferably takes place in an aqueous solutioncomposed of 2×SSPE and 0.1% SDS at room temperature for 10 minutes, thisbeing followed by another washing step in an aqueous solution composedof 2×SSPE and 0.1% SDS at 60° C., particularly preferably at 65° C., for15 minutes and by a final washing step with an aqueous solution composedof 0.4×SSPE and 0.02% SDS at 60° C., particularly preferably at 65° C.,for 15 minutes.

[0019] In a particularly preferred embodiment, a prehybridization iscarried out in an aqueous solution composed of 6×SSPE, 0.1% SDS and 5×Denhardt's reagent, and 500 μg/ml denatured herring sperm at 65° C. for3 hours. The hybridization takes place in an aqueous solution composedof 3×SSPE, 0.1% SDS, 5× Denhardt's reagent and 500 μg/ml denaturedherring sperm at 68° C. for 16 hours. The washing takes place in anaqueous solution composed of 2×SSPE and 0.1% SDS at 68° C. for 15minutes, this being followed by another washing step in an aqueoussolution composed of 1×SSPE and 0.1% SDS at 68° C. for 15 minutes and bya final washing step with an aqueous solution composed of 0.1×SSPE and0.1% SDS at 68° C. for 15 minutes.

[0020] The present invention also relates, of course, to modificationsof the aforementioned sequences, in particular those which display, bycomparision with the sequences shown in SEQ ID No. 1 or 3, nucleotideadditions, deletions, inversions, substitutions or the like, includingchemical derivatizations or replacement, exchange or addition of unusualnucleotides.

[0021] The invention also relates to nucleotide sequences which have adegree of homology of at least 80%, preferably 90%, to the sequencesshown in SEQ ID No. 1 or 3.

[0022] The nucleotide sequences of the present invention areadvantageous in particular inasmuch as they serve to modulate theexpression of a protein having the activity of a glutamine synthetase,in particular the GS-1 from sugarbeet. The nucleotide sequences of theinvention can be employed for altering, in particular reducing, and in aparticularly preferred manner completely suppressing, the amount ofglutamine synthetase formed, in particular the GS-1 from sugarbeet. In aparticularly preferred manner, the invention makes it possible throughmodulation of the expression in a tissue- and time-specific manner forthe deposition of glutamine in the storage organ of the sugarbeet to besuppressed without this inevitably entailing the need to employ tissue-and time-specific regulatory elements for the transgene, that is to saythe nucleotide sequence of the invention. This is because the nucleotidesequences of the invention encode the GS-1 of sugarbeet, which occursspecifically only in senescent leaves, and accordingly shows sitespecificity in relation to the expression in leaves and time specificityin relation to the expression during senescence. The nucleotidesequences of the invention make it possible to inhibit this glutaminesynthetase, which occurs specifically at leaf senescence, by means of asingle gene construct because the GS-1 of sugarbeet is a homooctamer andaccordingly the formation of all the GS-1 subunits can be switched offby means of a single gene construct. Accordingly, the invention providesin a particularly preferred manner for the use of antisense constructswhich specifically suppress the formation of protein, that is to sayGS-1, in the senescent leaves of sugarbeet. It is possible by use ofantisense constructs to inhibit the expression of the GS-1 of sugarbeetwhich occurs specifically at leaf senescence, so that glutamineformation and deposition of glutamine in the storage root is prevented.The growth of the sugarbeet is advantageously not impaired in thisprocess because GS-2 is not affected by the genetic manipulation of theplant cell.

[0023] In connection with the present invention, the activity of aprotein having the activity of a glutamine synthetase means an activityby which NH₄ ⁺ is bound enzymatically to C-4 of a glutamate moleculewith use of ATP.

[0024] In connection with the present invention, modulation of theexpression of a protein means a deliberate change, that is to sayincrease or reduction, achieved by genetic engineering methods, in theamount of protein in a cell compared with the amount of proteinnaturally present in the relevant cell at the relevant time.

[0025] The modulation of expression can take place by influencing thetranslation or transcription of the endogenous nucleotide sequenceencoding the protein, for example by introducing an antisense constructwhich partially or completely reduces the amount of translatable mRNA.An increase in the amount of protein may take place, for example, byintroducing a nucleotide sequence which encodes the protein and is underthe control of overexpressing regulatory elements, or by introducingmultiple gene copies.

[0026] Further alternative or additional modulations can be achieved byemploying tissue- or time-specific, inducible or constitutivelyexpressed regulatory elements which lead to an expression pattern whichis altered by comparison with the natural expression pattern in therelevant cell and at the relevant time or at the relevant stage ofdevelopment of the cell or the plant.

[0027] The present invention relates in a further embodiment to vectorscomprising at least one of the nucleotide sequences of the invention. Ina particularly preferred embodiment of the invention, such a vector isembodied as plasmid or viral vector.

[0028] The present invention also relates to vectors of theaforementioned type, where the at least one nucleotide sequence of thepresent invention is under the control of regulatory nucleotidesequences which are likewise present in the vector and which arearranged, for example, 5′, 3′, 5′ and 3′ or else within the nucleotidesequence. These regulatory nucleotide sequences may be heterologous tothe nucleotide sequence of the invention, that is to say be derived froma different organism or from a different gene, or homologous, that is tosay also naturally occurring together with the nucleotide sequences ofthe invention in a regulatory unit.

[0029] The invention accordingly also relates to vectors of theaforementioned type, where a nucleotide sequence controlling theexpression of the nucleotide sequence of the invention, in particular apromoter, is located 5′-wards of the nucleotide sequence of theinvention. In a particularly preferred embodiment of the invention, thepromoter is the 35 S promoter of CaMV or a promoter of the T-DNA ofAgrobacterium tumefaciens, for example the promoter of the nopalinesynthetase or octopine synthetase gene.

[0030] The invention provides in a further embodiment for atranscription termination unit, in particular a 3′-polyadenylationsignal, to be located 3′-wards of the nucleotide sequence of theinvention, particularly preferably the polyadenylation signal of the NOSgene of Agrobacterium tumefaciens.

[0031] The present invention provides in another preferred embodimentfor the regulatory sequences to be inducible, for example by externalfactors.

[0032] The invention provides in another preferred embodiment for theregulatory sequences of the expression of the nucleotide sequences ofthe invention controlled thereby to confer tissue specificity and/ortimespecificity, for example to bring about expression of an antisenseconstruct specifically in leaves during senescence.

[0033] The invention also provides for the nucleotide sequences of theinvention, where appropriate in a unit with the regulatory nucleotidesequences assigned to them, to be arranged in the vector together withnucleotide sequences which assist transfer and integration orrecombination of the nucleotide sequences of the invention whereappropriate with the regulatory nucleotide sequences assigned to theminto the genome of a transformed cell. The nucleotide sequences of theinvention can therefore be arranged, for example, between the left andright border region, flanked by only one border region in each caseand/or interrupted by one or more border regions of Agrobacteriumtumefaciens or Agrobacterium rhizogenes.

[0034] The present invention also relates to cells comprising at leastone of the aforementioned vectors. In a particularly preferred manner,such cells are bacterial cells, yeast cells or plant cells, inparticular plant cells from monocotyledonous or dicotyledonous plants,in particular sugarbeet. The present invention therefore particularlyrelates to a non-variety-specific cell of a plant which has beentransiently or stably transformed with a nucleotide sequence of theinvention, in particular which has this nucleotide sequence in itsgenome, for example in the form of an antisense construct. A plant meansa photosynthetically active organism including algae, mosses, ferns andhigher plants.

[0035] The invention also relates to cell assemblages, tissues, organs,parts of organs, cell cultures, calli, differentiated orundifferentiated cell aggregates, embryos, protoplasts etc. of anorganism which have, stably integrated into the genome or transientlypresent, at least one cell transformed with the nucleotide sequences ofthe invention. In a particularly preferred manner, the invention relatesto leaves, stalks, seeds, roots, storage organs, petals, flower organsetc. of a plant, said organs having at least one cell stably ortransiently transformed with the nucleotide sequences of the invention.In a particularly preferred manner, the plants or the parts thereof aretransformed in such a way that the transformed nucleotide sequence isstably inherited from generation to generation.

[0036] The present invention relates not only to cells, cellassemblages, calli and plant organs but also, of course, to plants, inparticular intact fertile plants which have been transformed by means ofthe nucleotide sequence of the invention and have in at least one oftheir cells at least one of these sequences, in particular stablyintegrated in their genome. The transformation preferably takes place,as stated hereinafter, nonbiologically, where agrobacterium-mediatedgene transfer is understood to be nonbiological. The resulting cells,and the plant tissues, plant organs, plant parts or plants having thesecells, are not variety-specific. On the contrary, the invention isapplicable to virtually all plants, plant families or plant genera.

[0037] In a particularly preferred manner, the transformed nucleotidesequence is heterologous to the transformed cell, that is to say is notnaturally present in the transformed cell, is not present in theartificially generated high copy number, or is not expressed at theplace or time at which it is expressed according to the invention. Incases in which the cell to be transformed already has an endogenousidentical or similar nucleotide sequence, the cell obtained by thetransformation of the invention differs from the initial cell forexample in that the introduced nucleotide sequence is present in adifferent genetic context in the genome, has different regulatoryelements, is arranged in antisense orientation to its regulatoryelements and/or is present in increased copy number.

[0038] The invention accordingly also relates to methods for producingtransgenic cells, where the nucleotide sequence of the invention to betransformed is introduced into the cell to be transformed by means of aconventional transformation method, for example microprojectilebombardment, agrobacterium-mediated gene transfer, electroporation,PEG-mediated transformation or the like.

[0039] The invention also relates to methods for producing transgenicplants having the nucleotide sequences of the invention, where cells orcell assemblages transformed with the nucleotide sequences of theinvention are cultivated and regenerated to intact, preferably fertile,plants. The cultivation and regeneration take place by conventionalmethods.

[0040] The invention also relates to a protein having the activity of aglutamine synthetase, in particular the GS-1 from sugarbeet, the latterbeing encoded by the nucleotide sequences of the invention, inparticular the nucleotide sequence shown in SEQ ID No. 1, particularlypreferably an amino acid sequence shown in SEQ ID No. 2. The inventionalso relates to proteins having the activity of a glutamine synthetase,in particular the GS-1 from sugarbeet, this sequence havingmodifications such as amino acid exchanges, deletions, additions,inversions or the like, and the protein having the activity of a GS-1from sugarbeet. The invention also relates to proteins which, at theamino acid level, have a degree of homology (identical amino acids) ofat least 90%, preferably 95%, with the sequence shown in SEQ ID No. 2.Proteins of this type can be prepared by employing the nucleotidesequences of the invention as cloning probes or hybridization probes foridentifying and cloning homologous genes encoding these proteins.

[0041] The invention relates in a further embodiment to monoclonal orpolyclonal antibodies against one of the aforementioned proteins, theseantibodies recognizing and binding one or more epitopes of saidproteins.

[0042] The invention relates in a further embodiment to methods foraltering glutamine metabolism in a sugarbeet, in particular formodulating the expression, particularly preferably for repressing, aprotein having the activity of a glutamine synthetase, in particular theGS-1 from sugarbeet, where the glutamine synthetase content of thesugarbeet is altered by transforming at least one sugarbeet cell with avector of the present invention, in particular transforming with avector having the nucleotide sequence of the invention in antisenseorientation, and regenerating a sugarbeet from the transformed cell oran association of cells. A sugarbeet generated in this way isadvantageously characterized in that the glutamine synthetase GS-1normally formed in its leaves during senescence is not formed becausethe expression of glutamine synthetase GS-1 is prevented because of theantisense construct present at least in the leaves and expressed there,so that the formation of glutamine in the leaves and, eventually, thedeposition of the glutamine in the storage root is prevented.

[0043] However, the invention also of course relates to methods foraltering glutamine metabolism in plants, in particular sugarbeet,according to which the glutamine synthetase content in particular cellsor organs is increased, where appropriate at certain times, inparticular by transforming gene constructs which make constitutiveand/or enhanced expression of the nucleotide sequences of the inventionpossible.

[0044] Further advantageous refinements of the invention are evidentfrom the dependent claims.

[0045] The invention is explained in detail by means of examples anddrawings belonging thereto.

[0046] SEQ ID No. 1 shows the translated region of the cDNA sequence ofthe GS-1 from sugarbeet.

[0047] SEQ ID No. 2 shows the amino acid sequence of the GS-1 fromsugarbeet.

[0048] SEQ ID No. 3 shows the complete cDNA sequence of the GS-1 fromsugarbeet.

[0049] The figures show:

[0050]FIG. 1 a Western blot of GS-1 obtained according to the invention,

[0051]FIG. 2 an autoradiogram of the Western blot in FIG. 1 and

[0052]FIG. 3 a diagrammatic depiction of the results obtained in FIGS. 1and 2.

EXAMPLE 1 Cloning of the cDNA for GS-1

[0053] Complete RNA was extracted from senescent sugarbeet leaves. Thiswas done by grinding 20 g of leaf material from senescent sugarbeetleaves in liquid nitrogen and transferring into 100 ml of uptake buffer(50 mM Tris-HCl pH 9.0, 100 mM NaCl, 10 mM EDTA, 2% w/v SDS and 0.2mg/ml proteinase K). This mixture was phenolized twice withphenol/chlorophorm/isoamyl alcohol (25/24/1), precipitated ({fraction(1/10)} volume of 3M NaAc, pH 6.5, one volume of isopropanol, 2 hours at−20° C.) and washed with 70% ethanol. After taking up in 10 ml of H₂O,10 μg/ml proteinase K and 2× precipitating with ¼ volume of 10 M LiCl at0° C. for 16 hours, the complete RNA was taken up in 2 ml of H₂O with 10μg/ml proteinase K. 5 mg of complete RNA were obtained.

[0054] Poly (A)⁺ mRNA was then isolated on an oligo-dT cellulose column.This was done by incubating 2 ml of complete RNA with 25 ml of bindingbuffer (400 mM NaCl, 10 mM Tris-HCl, pH 7.5 and 2% SDS) and oligo-dTcellulose (200 mg of oligo-dT cellulose) at room temperature for 30minutes while shaking gently. The mixture was transferred into a glasscolumn with cotton frit and washed dropwise with a washing buffer (100mM NaCl, 10 mM Tris-HCL, pH 7.5, 0.2% SDS) until the OD₂₆₀ was constantat 0.005. This was followed by elution with 10 ml of elution buffer (10mM Tris-HCL, pH 7.5) at 55° C. Precipitation was then carried out with{fraction (1/10)} volume of 3 M NaAc, pH 6.5 and 2 volumes of ethanol at−20° C. for two hours, and the mixture was taken up in 10 ml of bindingbuffer. The column purification was then repeated, the eluate wasphenolized before the precipitation, and the mRNA pellet was taken up inTE buffer. 50 μg of poly (A)+ mRNA were obtained.

[0055] cDNA was prepared using a cDNA synthesis kit from BoehringerMannheim in accordance with a standard protocol (5 μg of mRNA employed).The resulting cDNA was ligated into lambda vectors (NM 1149). 4 μg of NM1149 (EcoR I digested) and 0.2 μg of cDNA with EcoR I linkers wereemployed for this. After the ligation, the DNA was packaged in phages(NM 1149). The Gigapack® II gold packaging extract from Stratagene wasemployed for this in accordance with the standard protocol, using 4 μgof DNA and obtaining a titer of 140 000 pfu.

[0056] The resulting cDNA bank was screened, as was a cDNA bank fromsugarbeet root tissue (Lambda ZAP® II library, Stratagene, cDNA insertedfrom EcoR I and Not I into the Lambda ZAP® II vector system, titer: 250000 pfu, vector pBluescript® SK (−) with insertion isolated inaccordance with standard protocol by in vivo excision from Lambda ZAP®II) using a heterologous tobacco probe. The heterologous tobacco probeis described in Becker at al (1992) Plant. Molec. Biol. 19, 367-379. Forthe screening, E. coli bacteria were infected with the lambda phages andplated out. The phage DNA from lyzed bacteria was subsequentlytransferred to NC membranes (Plaquelift), and the membrane-bound DNA washybridized with a radiolabeled tobacco GS-1 cDNA probe.

[0057] The screening of the cDNA bank with the heterologous tobaccoprobe was carried out as follows. Firstly a prehybridization was carriedout with 6×SSPE, 0.1% SDS, 5× Denhardt's reagent and 500 μg/ml denaturedherring sperm at 61° C. for two hours. The hybridization was thencarried out at 61° C. for 16 hours with a solution of 3×SSPE, 0.1% SDS,5× Denhardt's reagent and 500 μg/ml denatured herring sperm. The washingwas carried out with 2×SSC and 0.1% SDS at 61° C. for 2×15 minutes. Awashing step was then carried out with 1×SSC and 0.1% SDS at 61° C. for15 minutes.

[0058] An autoradiogram of the membrane filters was developed, positivephages were isolated, and the corresponding DNA was extracted. The cDNAfound was subcloned into the plasmid pBluescript SK (Stratagene) andsequenced. The nucleotide sequence of the translated region of the cDNA,including the translation start codon ATG, is depicted in SEQ ID No. 1and has a length of 1068 bp. The complete sequence of the cDNA isdepicted in SEQ ID No. 3 and has a length of 1543 bp. The start codon islocated in position 199 to 201. The translated region terminates atposition 1266. A polyadenylation signal is located in the region ofnucleotides 1478 to 1508.

[0059] The amino acid sequence derived from SEQ ID No. 1 has a length of356 amino acids and is depicted in SEQ ID No. 2. The amino acid sequencerepresents the amino acid sequence of subunit P of the GS-1 fromsugarbeet. The protein is about 42 kDa in size and represents the onlysubunit of the GS-1 isoform which is present in the form of ahomooctamer in senescent sugarbeet leaves.

EXAMPLE 2 In Vitro Transcription and Translation of the P Subunit

[0060] The nucleotide sequence depicted in SEQ ID No. 1 was transcribedand translated in vitro. The cloned GS-1 DNA sequence employed for thiswas derived from the sugarbeet cDNA from root tissue mentioned inexample 1. In order to establish which GS-1 subunit this DNA sequencecodes for, an in vitro transcription and translation was carried outwith the “Linked in vitro SP 6/T7 Transcription/TranslationKit-radioactive” kit from Boehringer Mannheim. This was done byincubating 0.5 μl (0.5 μg) of plasmid DNA (pBluescript® SK (−) with theGS-1 insert), 5 μl of T7 transcription buffer and 14.5 μl of H₂O at 30°C. for 15 minutes. Then 10 μl of transcription reaction solution, 1.6 μlof ³⁵S-methionine and 38.4 μl of translation mix were incubated at 30°C. for 1 hour.

[0061] In addition, a protein extract was prepared from 5 g of sugarbeetleaves of various ages (in order to obtain all the GS-1 subunits forunambiguous identification). This extract was purified by FPLC, and thefractions with the highest GS-1 activities were concentrated. Proteinwas determined by the method of Bradford and revealed a proteinconcentration of about 1 μg/μl. Both this extract and the reactionmixture from the in vitro translation (with the radiolabeled GS-1protein) were mixed with the same volume of urea loading buffer. 20 μlof each of these were together put as sample for an isoelectric focusing(IEF) on an acrylamide capillary gel (1st dimension). Isoelectricfocusing took place at 190 V for 16 h.

[0062] Together with a protein size standard, the capillary gel wastransferred to an SDS gel in order to fractionate the proteins accordingto their size (2nd dimension). An SDS-PAGE took place at 140 V for 2 h.A Western blot (500 mA; 1 h) was prepared from this gel.

[0063] The nitrocellulose membrane was stained with Ponceau Red and thebands of the size standard were marked with a pencil.

[0064] After a blocking treatment (1 h), the membrane was incubated (16h; RT) with the 1st antibody (anti-GS; antibody against barley GS, RogerWallsgrove, Rothamsted Experimental Station, Harpenden, UK), 1:3 000 inblocking solution). The membrane was then washed 3× with TBS andincubated (3 h; RT) with the second antibody (1:2 000 in blockingsolution). After washing three more times, the color reaction with NBTand BCIP was effected by the alkaline phosphatase (10-20 min; 37° C.;dark). The dried membrane with the color-marked spots for the GS-1subunits was exposed to an X-ray film (exposure: 16 h; RT; dark).

[0065]FIG. 1 shows the membrane filter (Western blot) treated with GSantibodies and colored. The protein size standard is loaded on the left.The pH gradient in this case runs from pH 6 on the left to pH 4 on theright. The 4 spots for the GS-1 subunits are evident at the level of the43 kDa band (compare FIG. 3, although the sides are reversed in thiscase). The spot for the P subunit is marked (arrow).

[0066]FIG. 2 shows the autoradiogram of the membrane on which the bandsof the size standard are indicated on the left and a spot appears on theright at the level of the 43 kDa band. This spot was produced by theradiolabeled protein which had been transcribed and translated in vitro.It is possible by comparing the autoradiogram with the membrane toassign a spot on the membrane to the single spot on the film. This spotwas identified as subunit “P” (compare FIG. 3).

[0067]FIG. 3 represents a diagram of the proteins which can be labeledby GS antibodies. The direction of the pH gradient is indicated at thebottom (1st dimension) from pH 4 on the left to pH 6 on the right. Thebands of the size standard (2nd dimension) are depicted on the rightfrom 43 kDa at the top to 30 kDa at the bottom. Spots a, b, c and d,which are white in this diagram, show the positions of the GS-2subunits, which are likewise recognized by the antibody but can beseparated from GS-1 by FPLC. At the level of the 43 kDa band there arefour black spots s, i, p and w, which were identifiable on the basis oftheir size as the subunits forming the octamer of GS-1. The other blackspots u, v, x₁, x₂, y and z are possibly degradation products of the GSproteins.

EXAMPLE 3 Production of Transgenic Sugarbeet

[0068] A series of constructs each comprising a promoter which can beexpressed in plants, namely the CaMV 35 S promoter, each comprising asection of the sugarbeet GS-1 subunit gene of the invention in antisenseorientation, and each comprising the NOS terminator was produced. Thesections of the gene of the invention employed differed from oneanother. The gene cassettes produced in this way were ligated into thebinary vector (BIN19 with kanamycin resistance), and Agrobacteriumtumefaciens (with rifampicin resistance) was transformed with theresulting binary vectors by electroporation. The transformants underwentrifampicin and kanamycin selection. Subsequently, sugarbeet leaf disksand leaf stalks were transformed in a suspension with transformedagrobacteria, and callusing and shooting were induced with the planthormones NAA and BAP. After selection on kanamycin-containing medium andregeneration to intact plants by conventional protocols, it was possibleby means of measurements of the GS1 enzyme activities, SDS gelelectrophoreses, 2D PAGE and Northern blot analyses to demonstrate thatall the constructs employed, with the various gene sections, werepresent and active in the leaves of the transgenic, regenerated plant,and led to suppression of glutamine synthetase activity and formation insenescent sugarbeet leaves.

1. An isolated nucleotide sequence for reducing or preventing theexpression of a protein having the activity of a glutamine synthetase inthe senescing leaves of a transgenic plant selected from the groupconsisting of: a) the DNA sequence of SEQ ID Nos: 1 and 3; b) anucleotide sequence which encodes the amino acid sequence SEQ ID No: 2;c) a nucleotide sequence which is complementary to the nucleotidesequence of a) or b), and d) a nucleotide sequence which hybridizes witha nucleotide sequence of a) to c).
 2. A vector comprising the nucleotidesequence as claimed in claim
 1. 3. The vector as claimed in claim 2,where the vector is a plasmid or a viral vector.
 4. The vector asclaimed in claim 2, where the nucleotide sequence is operatively linkedto at least one regulatory nucleotide sequence.
 5. The vector as claimedin claim 4, where a promoter controlling the expression of thenucleotide sequence is arranged 5′-wards of the nucleotide sequence. 6.The vector as claimed in claim 4, where a 3′-polyadenylation signal isarranged 3′-wards of the nucleotide sequence.
 7. The vector as claimedin claim 4, where the regulatory sequence is inducible.
 8. The vector asclaimed in claim 4, where the regulatory sequence confers tissuespecificity and/or time specificity to the expression of the nucleotidesequence.
 9. The vector as claimed in claim 2, where the nucleotidesequence has antisense orientation to the promoter.
 10. A transgenicbacterial or plant cell comprising the vector as claimed in claim
 2. 11.The cell as claimed in claim 10, which is a sugarbeet cell.
 12. A plantcomprising at least one cell as claimed in claim
 10. 13. A seed of aplant, wherein said seed comprises at least one plant cell as claimed inclaim
 10. 14. A method for altering gluatmine metabolism in a sugarbeet,where synthesis of glutamine synthetase in senescing leaves in thesugarbeet is prevented or reduced by transforming at least one plantcell with the vector as claimed in claim 3, and regenerating thesugarbeet.
 15. A method for producing a transgenic sugarbeet which showsaltered glutamine metabolism, where the latter is based on a reductionof the content of glutamine synthetase in senescing leaves and where atleast one sugarbeet plant cell is transformed with the vector as claimedin claim 9, and the plant cell is regenerated to an intact plant.